Publications and other reference materials referred to herein, including reference cited therein, are incorporated herein by reference in their entirety and are numerically referenced in the following text and respectively grouped in the appended Bibliography which immediately precedes the claims.
Optical coherence tomography (OCT) is a 3D imaging method mainly associated with the production of high resolution cross sectional images of semi-transparent samples. The development of OCT and its applications to the biomedical optical imaging field are increasing every day. The main principles of OCT rely on light temporal coherence and interference, matter reflectivity and high sensitivity which are used to measure micro morphology of objects inside a turbid environment. Since its first appearance two decades ago [1], many technological schemes have been suggested to fulfill its goal [2]. However, despite their great diversity most OCT systems can be categorized into two main groups, time domain (TD) and frequency domain (FD) OCT. In principle, FD-OCT systems are faster, have higher signal to noise ratio (SNR) and better sensitivity [3], but hold lower resolution capabilities.
The key parameters of all OCT systems are resolvability (axial and lateral/transverse resolution), penetration capability, sensitivity and speed. Among all of the OCT techniques, the femto second pulse laser (fs-OCT) [4, 5] and time domain full field OCT systems (TD-FF-OCT) [6, 7] have been found to demonstrate the highest, sub-cellular, resolution capabilities.
Although state of the art fs-OCT systems demonstrate a higher sensitivity they are much more expensive and lack the extremely high lateral resolution and speed obtainable by TD-FF-OCT systems.
In most of the OCT modalities the axial resolution of the system is controlled by the source coherence length and, in some cases can be made as short as 1 μm [5, 7]. The lateral resolution of the system is controlled by the point spread function (PSF) of the focusing lens and theoretically can be made as high as ˜0.5 μm. The penetration capability is controlled mostly by the medium scattering and absorbing characteristics, but can be partially controlled by the central wavelength of the source; in some OCT systems it may be as great as ˜12 mm [9]. Due to the heterodyne natural amplification of the interferometer, the sensitivity can be extremely high, in some reported works as high as −110 dB [4], allowing detection of 10−11 of the incident optical power. The imaging speed in OCT is primarily controlled by the hardware and the imaging method used. In some recently reported works, 2.6×2.6×1.2 mm3 volumes were captured at 4 volumes per second, corresponding to ˜393 M pixels/s [10]. The above mentioned characteristics are what make this young imaging technique so appealing to so many different fields of research.
The main advantages of TD-FF-OCT are its extremely high 3D resolution, speed, simplicity and cost. In TD-FF-OCT the light source is comparatively cheap (halogen or xenon lamp) with very low coherence length. The method can use a high NA objective lens and therefore achieves very high resolution also in the transverse direction. The optical detection is made by a parallel detector CCD/CMOS (CCD—Charge Coupled Device; CMOS—Complementary Metal Oxide Semiconductor) camera so that enface images are obtained without parallel scanning the sample. The parallel detection simplifies the entire microscopy system and reduces production costs. TD-FF-OCT systems do not use any dynamic depth of focusing compensation techniques, as opposed to all other OCT methods. The TD-FF-OCT configuration allows real time en-face images, both interference and bright field; therefore, TD-FF-OCT systems may be used also as conventional optical microcopy system without additional optics.
However, in TD-FF-OCT the investigated sample is scanned by using a motorized linear stage, which is limited by speed and, like all mechanical systems, produces vibration noise. As in all OCT systems, the fact that wideband illumination is used to obtain high axial resolution forces the application of dispersion compensation techniques which in turn complicates the entire system architecture. In addition, when using a dry objective lens with high numerical aperture (NA), the interference signal contrast is severely eroded [11] and as a result the SNR and the sensitivity are reduced. Furthermore, the fact that the OCT signal extraction is done by mechanically actuating the reference mirror/objective limits both the accuracy and the lifetime of the microscope. Since traditional TD-FF-OCT systems use wideband illumination to achieve high axial resolution, true spectroscopic OCT imaging is not possible.
The time domain full field optical coherence tomography method, occasionally referred to as time domain full field optical coherence microscopy (TD-FF-OCM), is a rather new modality of OCT technology. Indeed, the first work on TD-FF-OCT was published only in 1998 [12], seven years after the concept of OCT was successfully demonstrated [1]. TD-FF-OCT is a more sophisticated version of the full field low coherence interference microscopy (FFLCIM), a method which is known for much longer time [13-16].
The first published work on FF-OCT [12] used the Taylor (sometime referred to as “Michelson”) interference microscopy configuration which in today's jargon is sometimes termed ‘wide field’ OCT (WF-OCT), for it holds a wider imaging field but a lower resolution capabilities. The axial resolution in [12] is governed by the source coherence length, which is 16 μm. The lateral resolution is determined by the objective lens NA and the CCD pixel size. The light emanating from the source (LED) is polarized, collimated and split at a polarized beam splitter. Two orthogonal field components are thus focused (by a 0.25 NA objective lens), one to the reference mirror and the other to the sample. By using a photo elastic modulator, a time dependent phase delay up is introduced between the two orthogonal signals. By successively grabbing four images with suitable phase difference the signal interference envelope is obtained. Due to the polarizer and the analyzer configuration, half of the reflected power is lost.
In 2002 the above method was employed with the Linnik interference microscope configuration, demonstrating much higher resolution capabilities [17-18]. In this work the optical light source was a halogen lamp with 260 nm Full Width at Half Maximum (FWHM) spectra centered at 840 nm wavelength. The wideband source allowed a 1.2 μm FWHM for the longitudinal resolution. The microscope objective lens NA was 0.3, providing a 1.3 μm lateral resolution at the center wavelength. The CCD camera operated in 200 fps and a piezoelectric ceramic material PZT (lead zirconate titanate (Pb[Zr(x)Ti(1−x)]O3)) was used for position modulating the reference arm at a rate of 50 Hz. Here, instead of using a photo elastic modulator, a PZT was used in order to demodulate the interference signal. It was reported, however, that the signal extraction was not ideal (fringes were still apparent). Also, due to the wideband spectra, the lateral resolution was somewhat limited as defocusing effects were stronger. As in all OCT systems with mechanically oscillating mirrors and continuous phase modulation, inertia problems and integration time drifts are also inherent here.
Wide field OCT (WF-OCT) was also demonstrated in high speed using a heterodyne detection scheme [19] by employing two CCD cameras (100 fps), however due to the long coherence length of the source SLD (Super Luminescent Diode) with 18 nm FWHM spectra centered at 826 nm wavelength) and the comparatively low NA objective lens (NA=0.14), both the lateral and the axial resolution were not very high (4 μm×17 μm, transverse×axial). In this technique the in phase (φ=0) and quadrature (φ=π/2) components of the interference signal are grabbed simultaneously at two different cameras (the phase delay is introduced via two liquid crystal (LC) shutters operating in π/2 delay). The DC level is known prior to image acquisition. Therefore real time enface imaging is obtained and the OCT cross section image is produced in 0.6 mm/s with measured 85 dB sensitivity. The concept of two cameras was also employed in ultrahigh resolution [20]; however, here as well, the demodulating means were mechanical. In general, using two cameras for high speed OCT is problematic as it means that a considerable effort must be put in order to align the two CCDs; a pixel to pixel alignment is required for accurate demodulation.
WF-OCT was also performed with real color by using three different color LEDs having their wavelength centered in the blue, green and red regions, 457.4 nm (FWHM=28.6 nm), 528.5 nm (FWHM=33.2 nm) and 638.4 nm (22.9 nm), respectively [21]. The envelope of the interference signal is extracted by position-modulating the reference mirror, thereby inducing four quadruplet signals (simple combination of the four signals results in the envelope signal). However, due to the large temporal coherence length of the source and the low objective lens NA, both the lateral and the axial resolution were not very high (15 μm×5 μm, transverse×axial). TD-FF-OCT was also applied with a rotatable polarizer phase shifter [22]. The phase shifter used to produce three images with three different phases by which the amplitude of the signal is extracted in similar fashion to the four quadruplet technique. Polarization sensitive OCT (PS-OCT) was also illustrated recently in the full field form [23]. In PS-FF-OCT, in addition to the traditional intensity en-face image representation a phase-retardation image is produced, which provides another level of information that may be useful to biological applications and clinician's observations.
The Fourier domain full field optical coherence tomography technique (FD-FF-OCT) is another interesting form of OCT technology which holds several advantages over the TD-FF-OCT. The FD-FF-OCT is a more sophisticated version of its predecessor the Fourier domain wide field optical coherence reflectometry, a method usually applied to surface profilometry [24]. Two major advantages are attributed to the FD form of FF-OCT: (i) it possesses very high SNR and (ii) it does not require any moving mechanical elements, neither for transverse nor for axial scanning.
However, its main disadvantages are (i) the requirements for frequency profile reshaping prior to Fourier transform (FT) operation, which slightly reduces the speed; (ii) twin image removal, which either limits the speed by elaborate computation or requires more sophisticated hardware; (iii) high extended depth of focus is required, which limits the lateral resolution; (iv) its comparatively narrowband tuning range, which limits the axial resolution; (v) its limited frequency spacing, which limits the optical imaging depth; and (vi) the requirement for having the entire wavelength image collection prior to OCT cross sectional image representation. Yet, the fact that in principle no moving parts are needed to perform 3D OCT imaging attracts a few groups of scientists to try and circumvent the above mentioned limitations.
Not many studies have been published on the FD-FF-OCT mode. As opposed to the conventional FD-OCT the FF form has only one applicable form. There are two fundamental techniques to perform FD-OCT, the spectral domain OCT (SD-OCT) and the swept source OCT (SS-OCT). In the case of FF-OCT, only the swept source configuration has been demonstrated (SS-FF-OCT).
The first, and one of the only high resolution, sub cellular, SS-FF-OCT works was published only in 2006 [25]. In this work a wide spectral source is obtained by pumping a Ti-Sapphire gain medium with a Nd:YAG laser. The wide gain bandwidth of the Ti-Sapphire laser produces a FWHM spectrum of 110 nm centered at 790 nm wavelength. The output spectrum is then tuned over almost the entire bandwidth with spectral spacing (line width) of 0.4 nm by a following acoustic optic modulator (AOM). The light is then coupled into multimode optical fiber (MMF) equipped with a suitable mode mixer which eliminates spackles. By controlling the wavelength transmitted by the AOM a plurality of ‘time encoded’ spectral images are obtained, then the inverse Fourier transform (IFT) operation is applied to the data stock and the OCT image is constructed. Due to the comparatively low NA of the objective lens and the moderate bandwidth of the spectrum used, OCT images of fruit fly eye were demonstrated with 1.3×1×0.2 mm3 volumes grabbed in 50 seconds and with axial by lateral resolution of 3 μm×4 μm. The fact that a femto second laser was used complicates the entire system and raises the costs. Also, this method does not overcome the problems of the twin image, the DC and coherent noises. As a result, only half of the potentially available imaging depth was employed.
SS-WF-OCT was also demonstrated for finger print detection, using Super-Luminescent Diode (SLD) in conjunction with AOM as the wavelength tuning device [26]. The SLD spectrum was only 48 nm FWHM and low NA projecting lens was used, therefore the lateral and axial resolution were not very high. A very similar work was also presented in [27]. SS-WF-OCT was very recently demonstrated in-vivo on a human retina [28] using a tunable light source with 850 nm center wavelength and effective FWHM of 25 nm with 0.05 nm line width. Both the lateral and the axial resolution were not very high (13 μm×13 μm) due to the low NA objectives and the comparatively narrow bandwidth of the source. Also in this work the problem of the twin image, the DC and the coherent noises were not overcome.
The subject of FD-FF-OCT is still a hot zone of research driven by the magical power of the Fourier transform analysis which permits 3D imaging without mechanically moving elements. However, for the time being, due to the requirement of large depth of field and the additional cost associated with the swept source this imaging modality is still, for high resolution applications, less favored than the TD-FF-OCT method.
In U.S. Pat. No. 6,940,602 (Dubois et al), a method and a system for obtaining interference images of an object are described. In this patent the interference signal is demodulated by mechanically oscillating one of the mirrors/objectives of the interference microscope. In this system the phase difference between the interference arms is changed sinusoidally and the interference signal is integrated along four time periods. By linear combination of these four phase images the phase and amplitude of the interference signal is obtained.
In U.S. Pat. No. 7,095,503 (Kim et al) a method and a system for obtaining true color OCT images using a wide field OCT (WF-OCT) configuration is depicted. The invention uses a color CCD parallel detector and a white light source. The true color OCT images are obtained by demodulating the reference arm and grabbing four phase shifted interference images of the object. The fact that the system uses the Taylor like interferometer configuration does not allow high lateral resolution images (˜15 μm). Also, since the CCD color bands are limited to a relatively narrow bandwidth, the axial resolution is limited to about 5 μm. This invention also describes an alternative light source; using three different color LEDs.
In U.S. Pat. No. 7,088,454 (Chang et al) a method and a system for extracting the OCT signal from 3 different images is described. The method is exemplified using a Taylor like interferometer with a super-luminescence diode. In this method, a first non interfering image is taken and then two interference images with π/2 phase difference are grabbed. A simple mathematical combination of these images produces the OCT images. The method uses mechanical modulation of the reference arm and is not capable of operating at high resolution due to the Taylor like configuration and the comparatively moderate bandwidth of the source.
In U.S. Pat. No. 7,950,801(Lacombe et al) a method and a system for demodulating the OCT signal of a FF-OCT system in real time are presented. The invention uses a Wollaston prism to project simultaneously four spatially resolved different interference images on the same parallel detector. Although the method is very fast it uses linearly polarized light to illuminate the sample, and therefore cannot be implemented on strongly birefringent samples. In addition, the fact that each interference image requires ¼th of the CCD sensor area limits the available field for imaging and reduces the effectiveness of the parallel detector. Furthermore, in this method the Wollaston prism is positioned just about the objective lens and therefore produces some aberration. Additionally, the four different images on the parallel detector must be matched with great precision.
In patent application number US 2009/0153876 (Chan et al) a system and a method for obtaining FF-OCT images in high speed are disclosed. The system comprises two separated cameras by means of which it extracts the OCT signal with only two measurements. The phase shift between these measurements is imposed by mechanically oscillating the reference mirror in similar fashion to the method described above in U.S. Pat. No. 6,940,602.
In U.S. Pat. No. 4,818,110 (Davidson et al.), a method and a system for obtaining high resolution images of microelectronic objects, established on the Linnik interference microscope configuration, is disclosed. In this invention the images are obtained based on the degree of coherence of the reference and sample images. To obtain the degree of coherence, two interference images are obtained with phase difference of π. Based on the fringe intensity (in the axial direction) the image is reconstructed.
In U.S. Pat. No. 5,194,918 (Kino et al), a method for extracting the envelope of an interference image is disclosed using a Mirau correlation microscope. The method uses the Fourier/Hilbert transform operation to separate the DC level from the carrier of the interference signal. After filtering the zero order, the inverse transform operation is applied which in turn yields the envelope of the carrier, i.e. the desired envelope. In this technique the entire space must be scanned before obtaining the full-field en-face images. The method requires a large number of sampling points for high accuracy. Also, when large fields are analyzed, the computation process is long. Therefore, this method is rather slow.
In U.S. Pat. No. 7,468,799 (de Groot et al), a method of obtaining the interference image pattern of a thin film and of microelectronic samples is disclosed. The method is demonstrated using the Mirau and the Linnik scanning interferometers and is established on the fringe pattern obtained by axially scanning the sample, no demodulating means are described.
In patent application number US 2011/0181888A1 from Jul. 28, 2011, a Fourier domain wide field optical coherence tomography system with an extended depth of field is disclosed. The invention uses phase masks such as an annular phase mask which produces a π/2 phase shift on light passing the annular region. The mask is positioned in at least one of the microscope interference arms. Optionally the phase mask is positioned on one of the surfaces of the objective lens. Although the invention is applicable to OCT in general it mainly describes a wide field OCT (WF-OCT) system. This configuration allows only comparatively low resolution.